Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.     

Analysis of the antigen binding site of anti-deoxycholate monoclonal antibody using a novel affinity labeling reagent, acyl adenylate

Large-scale analysis of protein-protein interaction sites is especially needed in the postgenomic era. The combination of affinity labeling with mass spectrometry is a potentially useful high-throughput screening method for this purpose. However, reagents in current use are not ideal as some cause damage to the target molecule and others have poor solubility in physiologic aqueous buffers. In this paper, we describe a novel affinity labeling reagent, acyl adenylate, which is highly soluble in aqueous solutions and reacts in a pH-dependent manner. The adenylate of deoxycholic acid reacts with amino groups on the side chain of a lysine residue and at the N-terminus of proteins/peptides. The reactivity and stability of this reagent were investigated, and it was confirmed that, after formation of a reversible ligand-protein complex under weakly acidic conditions, derivatization with acyl adenylate occurred at the target site under weakly alkaline condition. We further demonstrated the utility of this reagent for affinity labeling using a monoclonal antibody with high affinity for deoxycholic acid. Competitive ELISA indicated that deoxycholic acid was labeled around the antibody ligand binding site, thus enabling the structural elucidation of the ligand-protein interaction. In addition, LC/ESI-MS/MS analysis of the labeled peptide obtained by enzymatic digestion and affinity extraction allowed the identification of the structure surrounding the antigen binding site.     

A nonenzymatic modification of the amino-terminal domain of histone H3 by bile acid acyl adenylate

Although it has been proposed that the secondary bile acids, deoxycholic acid and lithocholic acid, increase the number of aberrant crypt foci in the colon and may act as colon tumor promoters, there is little evidence detailing their mechanism of action. Histones play an important role in controlling gene expression, and the posttranslational modification of histones plays a role in regulation of intracellular signal transduction. In particular, the amino-terminal tail domain of histone H3 is sensitive to several posttranslational modifications, and acetylation of this domain changes its electrostatic environment and results in the loss of native folding. Therefore, we studied the modification of epsilon-amino groups on human histone H3 by deoxycholyl adenylate, which is an active intermediate in deoxycholyl thioester biosynthesis. After incubation of recombinant human histone H3 with a smaller amount of acyl adenylate, followed by enzymatic digestion, the peptide fragment mixtures were analyzed by matrix-assisted laser desorption ionization mass spectrometry. These data showed the formation of only one adduct fragment, which corresponded to amino acids 3-8 with a deoxycholate adduct, suggesting that the epsilon-amino group of Lys(4) had the highest reactivity. This novel modification, formation of a bile acid adduct on the histone H3 amino-terminal tail domain through an active acyl adenylate, may relate to the carcinogenesis-promoting effects of secondary bile acids.     

Signals from intra-abdominal fat modulate insulin and leptin sensitivity through different mechanisms: neuronal involvement in food-intake regulation

Intra-abdominal fat accumulation is involved in development of the metabolic syndrome, which is associated with insulin and leptin resistance. We show here that ectopic expression of very low levels of uncoupling protein 1 (UCP1) in epididymal fat (Epi) reverses both insulin and leptin resistance. UCP1 expression in Epi improved glucose tolerance and decreased food intake in both diet-induced and genetically obese mouse models. In contrast, UCP1 expression in Epi of leptin-receptor mutant mice did not alter food intake, though it significantly decreased blood glucose and insulin levels. Thus, hypophagia induction requires a leptin signal, while the improved insulin sensitivity appears to be leptin independent. In wild-type mice, local-nerve dissection in the epididymis or pharmacological afferent blockade blunted the decrease in food intake, suggesting that afferent-nerve signals from intra-abdominal fat tissue regulate food intake by modulating hypothalamic leptin sensitivity. These novel signals are potential therapeutic targets for the metabolic syndrome.     

Identification of a novel bile acid in swans, tree ducks, and geese: 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid

By HPLC, a taurine-conjugated bile acid with a retention time different from that of taurocholate was found to be present in the bile of the black-necked swan, Cygnus melanocoryphus. The bile acid was isolated and its structure, established by (1)H and (13)C NMR and mass spectrometry, was that of the taurine N-acyl amidate of 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid. The compound was shown to have chromatographic and spectroscopic properties that were identical to those of the taurine conjugate of authentic 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid, previously synthesized by us from ursodeoxycholic acid. By HPLC, the taurine conjugate of 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid was found to be present in 6 of 6 species in the subfamily Dendrocygninae (tree ducks) and in 10 of 13 species in the subfamily Anserinae (swans and geese) but not in other subfamilies in the Anatidae family. It was also not present in species from the other two families of the order Anseriformes. 3alpha,7alpha,15alpha-Trihydroxy-5beta-cholan-24-oic acid is a new primary bile acid that is present in the biliary bile acids of swans, tree ducks, and geese and may be termed 15alpha-hydroxy-chenodeoxycholic acid.     

Microbial production of optically active β-phenylalanine ethyl ester through stereoselective hydrolysis of racemic β-phenylalanine ethyl ester

The ability to produce (R)- or (S)-β-phenylalanine ethyl ester (3-amino-3-phenylpropionic acid ethyl ester, BPAE) from racemic BPAE through stereoselective hydrolysis was screened for in BPAE-assimilating microorganisms. Sphingobacterium sp. 238C5 and Arthrobacter sp. 219D2 were found to be potential catalysts for (R)- and (S)-BPAE production, respectively. On a 24-h reaction, with 2.5% (w/v) racemic BPAE (130 mM) as the substrate and wet cells of Sphingobacterium sp. 238C5 as the catalyst, 1.15% (w/v) (R)-BPAE (60 mM) with enantiomeric purity of 99% e.e. was obtained, the molar yield as to racemic BPAE being 46%. On a 48-h reaction, with 2.5% (w/v) racemic BPAE (130 mM) as the substrate and wet cells of Arthrobacter sp. 219D2 as the catalyst, 0.87% (w/v) (S)-BPAE (45 mM) with enantiomeric purity of 99% e.e. was obtained, the molar yield as to racemic BPAE being 35%. The enzyme stereoselectively hydrolyzing (S)-BPAE was purified to homogeneity from the cell-free extract of Sphingobacterium sp. 238C5. The enzyme was a monomeric protein with a molecular mass of about 42,000. The enzyme catalyzed hydrolysis of β-phenylalanine esters, while the common aliphatic and aromatic carboxylate esters were not catalyzed.     

Characteristic features in the structure and collagen-binding ability of a thermophilic collagenolytic protease from the thermophile Geobacillus collagenovorans MO-1

A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, extracellularly produces a collagenolytic protease with a large molecular mass. Complete nucleotide sequencing of this gene after gene cloning revealed that the collagenolytic protease is a member of the subtilisin family of serine proteases and consists of a signal sequence for secretion, a prosequence for maturation, a catalytic region, 14 direct repeats of 20 amino acids at the C terminus, and a region with unknown function intervening between the catalytic region and the numerous repeats. Since the unusual repeats are most likely to be cleaved in the secreted form of the enzyme, the intervening region was investigated to determine whether it participates in collagen binding to facilitate collagen degradation. It was found that the mature collagenolytic protease containing the intervening region at the C terminus bound collagen but not the other insoluble proteins, elastin and keratin. Furthermore, the intervening region fused with glutathione S-transferase showed a collagen-binding ability comparable to that of the mature collagenolytic protease. The collagen-binding ability was finally attributed to two-thirds of the intervening region which is rich in beta-strands and is approximately 35 kDa in molecular mass. In the collagenolytic protease from strain MO-1, hydrogen bonds most likely predominate over the hydrophobic interaction for collagen binding, since a higher concentration of NaCl released collagen from the enzyme surface but a nonionic detergent could not. To the best of our knowledge, this is the first report of a thermophilic collagenolytic protease containing the collagen-binding segment.     

Redox potentials of the blue copper sites of bilirubin oxidases

The redox potentials of the multicopper redox enzyme bilirubin oxidase (BOD) from two organisms were determined by mediated and direct spectroelectrochemistry. The potential of the T1 site of BOD from the fungus Myrothecium verrucaria was close to 670 mV, whereas that from Trachyderma tsunodae was >650 mV vs. NHE. For the first time, direct electron transfer was observed between gold electrodes and BODs. The redox potentials of the T2 sites of both BODs were near 390 mV vs. NHE, consistent with previous finding for laccase and suggesting that the redox potentials of the T2 copper sites of most blue multicopper oxidases are similar, about 400 mV.     

Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide-membrane interactions

In face of accumulated reports demonstrating that uptake of some cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of cell fixation artifacts, we conducted a systematic analysis on the mechanism responsible for the cellular uptake of the S4(13)-PV karyophilic cell-penetrating peptide. The results reviewed here show that the S4(13)-PV peptide is able to very efficiently accumulate inside live cells in a rapid, non-toxic and dose-dependent manner, through a mechanism distinct from endocytosis. Comparative analysis of peptide uptake by mutant cells lacking heparan sulfate proteoglycans demonstrates that, although not mandatory, their presence at cell surface facilitates the cellular uptake of the S4(13)-PV peptide. Furthermore, we demonstrate that upon interaction with lipid vesicles, the S4(13)-PV peptide undergoes significant conformational changes that are consistent with the formation of helical structures. Such conformational changes occur concomitantly with a penetration of the peptide into the lipid bilayer, strongly suggesting that the resulting helical structures are crucial for the non-endocytic cellular uptake of the S4(13)-PV peptide. Overall, our data support that, rather than endocytosis, the cellular uptake of the S4(13)-PV cell-penetrating peptide is a consequence of its direct translocation through cell membranes following conformational changes induced by peptide-membrane interactions.     

Crohn's disease risk alleles on the NOD2 locus have been maintained by natural selection on standing variation

Risk alleles for complex diseases are widely spread throughout human populations. However, little is known about the geographic distribution and frequencies of risk alleles, which may contribute to differences in disease susceptibility and prevalence among populations. Here, we focus on Crohn's disease (CD) as a model for the evolutionary study of complex disease alleles. Recent genome-wide association studies and classical linkage analyses have identified more than 70 susceptible genomic regions for CD in Europeans, but only a few have been confirmed in non-European populations. Our analysis of eight European-specific susceptibility genes using HapMap data shows that at the NOD2 locus the CD-risk alleles are linked with a haplotype specific to CEU at a frequency that is significantly higher compared with the entire genome. We subsequently examined nine global populations and found that the CD-risk alleles spread through hitchhiking with a high-frequency haplotype (H1) exclusive to Europeans. To examine the neutrality of NOD2, we performed phylogenetic network analyses, coalescent simulation, protein structural prediction, characterization of mutation patterns, and estimations of population growth and time to most recent common ancestor (TMRCA). We found that while H1 was significantly prevalent in European populations, the H1 TMRCA predated human migration out of Africa. H1 is likely to have undergone negative selection because 1) the root of H1 genealogy is defined by a preexisting amino acid substitution that causes serious conformational changes to the NOD2 protein, 2) the haplotype has almost become extinct in Africa, and 3) the haplotype has not been affected by the recent European expansion reflected in the other haplotypes. Nevertheless, H1 has survived in European populations, suggesting that the haplotype is advantageous to this group. We propose that several CD-risk alleles, which destabilize and disrupt the NOD2 protein, have been maintained by natural selection on standing variation because the deleterious haplotype of NOD2 is advantageous in diploid individuals due to heterozygote advantage and/or intergenic interactions.